CCK-related (CCK, gastrin) and VIP-related peptides [VIP-related peptides [VIP, PACAP] have widespread effects in the gastrointestinal tract (GI) and central nervous system affecting such diverse functions as motility, secretion and growth of normal and neoplastic tissues. The actions of CCK-related peptides are mediated by two receptors (CCKA-R, CCKB-R) and VIP-PACAP-related peptides by three receptors (VIP-1-R, VIP-2-R, PACAP-R). Studies in this project are aimed at understanding the molecular basis of action of these different receptors. In general, three groups of studies have been performed in this project. To define the molecular pharmacology of VIP receptors, the rat and human VIP-1-R and VIP-2-R receptors (R. Jensen) are being cloned and stably transfected in CHO and PANC1 cells. Furthermore, various native cells are being screened by RT-PCR, Northern blots and binding studies to identify cell lines that have only one native VIP-R subtype. The VIP pharmacophore is being determined for each VIP-R subtype by alanine and D-amino acid screening, to attempt to identify selective ligands. In a second study (S. Wank), the relationship of VIP and GLP-1 to the peptide helodermin and exendin-4 was investigated. To determine whether mammalian homologues of these two peptides existed, their DNA's were cloned from Gila-monster salivary glands, as well as those for G-monster VIP, PHI, PACAP and GLP-1. Probing of rat and human tissues by Northern and Southern blots failed to identify helodermin or exendin-4 specific cDNAs and suggests they are not present in mammals. In a third study (S. Wank), the role of the extracellular domains of CCK receptors (CCK-R) in agonist binding was investigated. Using site-directed mutagenesis and CCK-A-R and CCK-B-R chimeric receptors it was found that CCK-8 interacts at multiple contact points in the extracellular domains and that CCK-A-R and CCK-B-R have distinct binding sites despite their shared high affinity for CCK-8.